AAV--A NOVEL VECTOR FOR GLOBIN GENE THERAPY

基因治疗的伟大愿望之一是最终开发出 技术将提供一种可行的方法来纠正基因 缺陷和防治传染病。我的实验室正在从事 人细小病毒缺陷型腺病毒的分子生物学研究 解决病毒(AAV)，希望开发一种安全、高效的病毒 用于人类基因治疗的载体。 AAV是一种依赖型细小病毒。也就是说，它需要与 另一种病毒(腺病毒或疱疹病毒的某些成员 组)，以便在培养细胞中进行生产性感染。在……里面 作为一种裂解性感染，AAV DNA以4.7千碱基双链形式复制 分子，并被包装成病毒粒子，作为线形单链 两极。在没有与辅助病毒重叠感染的情况下，AAV 基因组通过其末端整合到特定位点的宿主基因组中 并以潜伏状态存在，直到细胞被感染 有帮手病毒。然后AAV DNA被“拯救”，复制并 建立了正常的生殖性感染。AAV的宿主范围很广 传染性(人、猴子、老鼠等)当与该病毒同时感染时 合适的帮手。事实上，与目前的逆转录病毒载体相比 AAV的这些功能对于将AAV用作 病毒载体。人类AAV有许多优势。其中一些是： (1)它在人类中普遍存在，(2)AAV可以浓缩到滴度 每毫升超过10(9)个感染单位，以及(3) 完全无致病性的整合病毒。正在进行的研究是 揭示了这种非致病性人类病毒现在可以用于 以载体的形式利用。 我们已经开发了一种包装系统，它允许高效 将外源基因包裹到AAV病毒粒子中。我们还确认了 有效整合所需的基本顺式作用序列 转化成宿主细胞的DNA。最后，我们描述了野生型整合 并揭示了特定地点整合的令人兴奋的结果。这 最后的观察清楚地表明，AAV是一种真核病毒载体， 这是人类基因治疗的潜力所在。《公约》的总体目标 建议的工作是充分检验AAV作为具体应用的可行性 转导病毒载体用于珠蛋白基因治疗。

One of the great aspirations of gene therapy is to eventually develop technology which will provide a feasible approach to correct genetic defects and combat infectious diseases. My laboratory is engaged in studying the molecular biology of the defective human parvovirus adeno- addressed virus (AAV) in hopes of developing a safe efficient viral vector for human gene therapy. AAV is a dependent parvovirus. That is, it requires coinfection with another virus (either adenovirus or certain members of the herpes virus group) in order to undergo a productive infection in cultured cells. In a lytic infection, AAV DNA replicates as a 4.7 kilobase double-stranded molecule and is packaged into virion as linear single strands of both polarities. In the absence of coinfection with helper virus, the AAV genome integrates via its ends into the host genome in a site specific manner and resides there in a latent state until the cell is infected with helper virus. Then the AAV DNA is "rescued", replicates and establishes a normal productive infection. AAV has a broad host range for infectivity (human, monkey, mouse, etc.) when coinfected with the appropriate helper. In fact, compared to the current retroviral vectors these features of AAV are of considerable interest in utilizing AAV as a viral vector. Human AAV has a number of advantages. Some of them are: (1) it is ubiquitous in humans, (2) AAV can be concentrated to titers exceeding 10(9) infectious units per milliliter, and (3) it is completely nonpathogenic integrating virus. Ongoing research is revealing that this nonpathogenic human virus is now accessible for utilization as a vector. We have developed a packaging system which allows for efficient encapsidation of foreign genes into AAV virions. We have also identified the essential cis-acting sequences required for efficient integration into host cell DNA. Finally, we have characterized wild type integration and uncovered the exciting result of site-specific integration. This last observation clearly sets apart AAV as a eucaryotic viral vector and it's potential for gene therapy in humans. The overall objective of the proposed work is to fully test the feasibility of AAV as a specific transducing viral vector for globin gene therapy.